Identification of the TRM2 gene encoding the tRNA(m5U54)methyltransferase of Saccharomyces cerevisiae

The presence of 5-methyluridine (m5U) at position 54 is a ubiquitous feature of most bacterial and eukaryotic elongator tRNAs. In this study, we have identified and characterized the TRM2 gene that encodes the tRNA(m5U54)methyltransferase, responsible for the formation of this modified nucleoside in Saccharomyces cerevisiae. Transfer RNA isolated from TRM2-disrupted yeast strains does not contain the m5U54 nucleoside. Moreover, a glutathione S-transferase (GST) tagged recombinant, Trm2p, expressed in Escherichia coli displayed tRNA(m5U54)methyltransferase activity using as substrate tRNA isolated from a trm2 mutant strain, but not tRNA isolated from a TRM2 wild-type strain. In contrast to what is found for the tRNA(m5U54)methyltransferase encoding gene trmA+ in E. coli, the TRM2 gene is not essential for cell viability and a deletion strain shows no obvious phenotype. Surprisingly, we found that the TRM2 gene was previously identified as the RNC1/NUD1 gene, believed to encode the yNucR endo-exonuclease. The expression and activity of the yNucR endo-exonuclease is dependent on the RAD52 gene, and does not respond to increased gene dosage of the RNC1/NUD1 gene. In contrast, we find that the expression of a trm2-LacZ fusion and the activity of the tRNA(m5U54)methyltransferase is not regulated by the RAD52 gene and does respond on increased gene dosage of the TRM2 (RNC1/NUD1) gene. Furthermore, there was no nuclease activity associated with a GST-Trm2 recombinant protein. The purified yNucR endo-exonuclease has been reported to have an NH2-D-E-K-N-L motif, which is not found in the Trm2p. Therefore, we suggest that the yNucR endo-exonuclease is encoded by a gene other than TRM2.     

The T-arm of tRNA is a substrate for tRNA (m5U54)-methyltransferase

Fragments of Escherichia coli FUra-tRNA(1Val) as small as 15 nucleotides form covalent complexes with tRNA (m5U54)-methyltransferase (RUMT). The sequence essential for binding includes position 52 of the T-stem and the T-loop and extends toward the 3' acceptor end of FUra-tRNA. The in vitro synthesized 17mer T-arm of E. coli tRNA(1Val), composed of the seven-base T-loop and 5-base-pair stem, is a good substrate for RUMT. The Km is decreased 5-fold and kcat is decreased 2-fold compared to the entire tRNA. The T-arm structure could be further reduced to an 11mer containing the loop and two base pairs and still retain activity; the Km was similar to that of the 17mer T-arm, whereas kcat was decreased an additional 20-fold. The data indicate that the primary specificity determinants for the RUMT-tRNA interaction are contained within the primary and secondary structure of the T-arm of tRNA.     

Amino acid residues of the Escherichia coli tRNA(m5U54)methyltransferase (TrmA) critical for stability, covalent binding of tRNA and enzymatic activity

The Escherichia coli trmA gene encodes the tRNA(m⁵U54)methyltransferase, which catalyses the formation of m⁵U54 in tRNA. During the synthesis of m⁵U54, a covalent 62-kDa TrmA-tRNA intermediate is formed between the amino acid C324 of the enzyme and the 6-carbon of uracil. We have analysed the formation of this TrmA-tRNA intermediate and m⁵U54 in vivo, using mutants with altered TrmA. We show that the amino acids F188, Q190, G220, D299, R302, C324 and E358, conserved in the C-terminal catalytic domain of several RNA(m⁵U)methyltransferases of the COG2265 family, are important for the formation of the TrmA-tRNA intermediate and/or the enzymatic activity. These amino acids seem to have the same function as the ones present in the catalytic domain of RumA, whose structure is known, and which catalyses the formation of m⁵U in position 1939 of E. coli 23S rRNA. We propose that the unusually high in vivo level of the TrmA-tRNA intermediate in wild-type cells may be due to a suboptimal cellular concentration of SAM, which is required to resolve this intermediate. Our results are consistent with the modular evolution of RNA(m⁵U)methyltransferases, in which the specificity of the enzymatic reaction is achieved by combining the conserved catalytic domain with different RNA-binding domains.     

Covalent adducts between tRNA (m5U54)-methyltransferase and RNA substrates.

The interaction of tRNA (m5U54)-methyltransferase (RUMT) with in vitro synthesized unmodified tRNA and a 17-base oligoribonucleotide analog of the T-arm of tRNA in the absence of AdoMet has been investigated. Binary complexes are formed which are isolable on nitrocellulose filters and are composed of noncovalent and covalent complexes in nearly equal amounts. The covalent RUMT-RNA complexes are stable to SDS-PAGE and migrate slower than free enzyme or RNA. Kinetic and thermodynamic constants involved in formation and disruption of noncovalent and covalent binary complexes have been determined and interpreted in the context of steady-state kinetic parameters of the enzyme-catalyzed methylation and 5-H exchange of substrate. The results show that the isolable covalent complex is kinetically incompetent as an intermediate for methylation. Isotope trapping experiments show that when AdoMet is added to preformed binary complex, all bound tRNA is converted to methylated product; thus, the covalent complexes are chemically competent to form products. We have concluded that, after a reversible binary complex is formed, the catalytic thiol adds to the 6-carbon of the U54 of tRNA. The initial adduct leaves the reaction pathway to protonation at carbon 5; the latter can deprotonate and re-enter the pathway to form methylated product. It is speculated that covalent binary RUMT-RNA adducts may serve as depots of enzyme-tRNA complexes primed for methylation, or in unknown roles with RNAs other than tRNA.     

Enzymatic mechanism of tRNA (mU54)methyltransferase

tRNA (m⁵U54)methyltransferase (RUMT) catalyzes the methylation of uridine 54 of transfer RNA by S-adenosyl-l-methionine. In this report, we present the enzymatic mechanism of RUMT, including the stereochemical course of the methylation reaction, and discuss RUMT-tRNA recognition. As part of its enzymatic mechanism, we postulate that RUMT catalyzes the disruption of RNA-RNA contacts. We also show that many nucleotide substitutions can be made in the T-loop of tRNA without affecting RUMT binding, indicating that the recognition of the T-loop by RUMT is not stringent.     

Stereochemistry of methyl transfer catalyzed by tRNA (m5U54)-methyltransferase--evidence for a single displacement mechanism.

tRNA (m5U54)-methyltransferase (RUMT) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the 5-carbon of uridine 54 of tRNA. We have determined the steric course of methyl transfer, using (methyl-R)- and (methyl-S)-[methyl-2H1,3H]-AdoMet as the chiral methyl donors, and tRNA lacking the 5-methyl group at position 54 as the acceptor. Following methyl transfer, ribothymidine was isolated and degraded to chiral acetic acid for configurational analysis. Transfer of the chiral methyl group to U54 proceeded with inversion of configuration of the chiral methyl group, suggesting that RUMT catalyzed methyl transfer occurs by a single SN2 displacement mechanism.     

The carboxyl-terminal extension of yeast tRNA m5C methyltransferase enhances the catalytic efficiency of the amino-terminal domain

The human tRNA m(5)C methyltransferase is a potential target for anticancer drugs because it is a novel downstream target of the proto-oncogene myc, mediating Myc-induced cell proliferation. Sequence comparisons of RNA m(5)C methyltransferases indicate that the eukaryotic enzymes possess, in addition to a conserved catalytic domain, a large characteristic carboxyl-terminal extension. To gain insight into the function of this additional domain, the modular architecture of the yeast tRNA m(5)C methyltransferase orthologue, Trm4p, was studied. The yeast enzyme catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to carbon 5 of cytosine at different positions depending on the tRNAs. By limited proteolysis, Trm4p was shown to be composed of two domains that have been separately produced and purified. Here we demonstrate that the aminoterminal domain, encompassing the active site, binds tRNA with similar affinity as the whole enzyme but shows low catalytic efficiency. The carboxyl-terminal domain displays only weak affinity for tRNA. It is not required for m(5)C formation and does not appear to contribute to substrate specificity. However, it enhances considerably the catalytic efficiency of the amino-terminal domain.     

Distinct origins of tRNA(m1G37) methyltransferase

The enzyme tRNA(m1G37) methyltransferase catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to the N1 position of G37 in the anticodon loop of a subset of tRNA. The modified guanosine is 3' to the anticodon and is important for maintenance of reading frame during decoding of genetic information. While the methyltransferase is well conserved in bacteria and is easily identified (encoded by the trmD gene), the identity of the enzyme in eukarya and archaea is less clear. Here, we report that the enzyme encoded by Mj0883 of Methanocaldococcus jannaschii is the archaeal counterpart of the bacterial TrmD. However, despite catalyzing the same reaction and displaying similar enzymatic properties, MJ0883 and bacterial TrmD are completely unrelated in sequence. The catalytic domain of MJ0883, when aligned with the five known structural folds (I-V) that have been described to bind AdoMet, is of the class I fold, similar to the ancient Rossmann fold that binds nucleotides. In contrast, the catalytic domain of the bacterial TrmD has the unusual class IV fold of a trefoil knot structure. Thus, both the sequence and structural arrangements of tRNA(m1G37) methyltransferase have distinct evolutionary origins among primary kingdoms, revealing an unexpected but remarkable non-orthologous gene displacement to achieve an important tRNA modification.     

Substrate tRNA recognition mechanism of tRNA (m7G46) methyltransferase from Aquifex aeolicus

Transfer RNA (m7G46) methyltransferase catalyzes the methyl transfer from S-adenosylmethionine to N7 atom of the guanine 46 residue in tRNA. Analysis of the Aquifex aeolicus genome revealed one candidate open reading frame, aq065, encoding this gene. The aq065 protein was expressed in Escherichia coli and purified to homogeneity on 15% SDS-polyacrylamide gel electrophoresis. Although the overall amino acid sequence of the aq065 protein differs considerably from that of E. coli YggH, the purified aq065 protein possessed a tRNA (m7G46) methyltransferase activity. The modified nucleoside and its location were determined by liquid chromatography-mass spectroscopy. To clarify the RNA recognition mechanism of the enzyme, we investigated the methyl transfer activity to 28 variants of yeast tRNAPhe and E. coli tRNAThr. It was confirmed that 5'-leader and 3'-trailer RNAs of tRNA precursor are not required for the methyl transfer. We found that the enzyme specificity was critically dependent on the size of the variable loop. Experiments using truncated variants showed that the variable loop sequence inserted between two stems is recognized as a substrate, and the most important recognition site is contained within the T stem. These results indicate that the L-shaped tRNA structure is not required for methyl acceptance activity. It was also found that nucleotide substitutions around G46 in three-dimensional core decrease the activity.     

Crystal structure of tRNA(m1G37)methyltransferase: insights into tRNA recognition

tRNA(m(1)G37)methyltransferase (TrmD) catalyzes the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to G(37) within a subset of bacterial tRNA species, which have a G residue at the 36th position. The modified guanosine is adjacent to and 3' of the anticodon and is essential for the maintenance of the correct reading frame during translation. Here we report four crystal structures of TrmD from Haemophilus influenzae, as binary complexes with either AdoMet or S-adenosyl-L-homocysteine (AdoHcy), as a ternary complex with AdoHcy and phosphate, and as an apo form. This first structure of TrmD indicates that it functions as a dimer. It also suggests the binding mode of G(36)G(37) in the active site of TrmD and the catalytic mechanism. The N-terminal domain has a trefoil knot, in which AdoMet or AdoHcy is bound in a novel, bent conformation. The C-terminal domain shows structural similarity to trp repressor. We propose a plausible model for the TrmD(2)-tRNA(2) complex, which provides insights into recognition of the general tRNA structure by TrmD.     

Sequence-structure-function studies of tRNA:m5C methyltransferase Trm4p and its relationship to DNA:m5C and RNA:m5U methyltransferases

Three types of methyltransferases (MTases) generate 5-methylpyrimidine in nucleic acids, forming m5U in RNA, m5C in RNA and m5C in DNA. The DNA:m5C MTases have been extensively studied by crystallographic, biophysical, biochemical and computational methods. On the other hand, the sequence-structure-function relationships of RNA:m5C MTases remain obscure, as do the potential evolutionary relationships between the three types of 5-methylpyrimidine-generating enzymes. Sequence analyses and homology modeling of the yeast tRNA:m5C MTase Trm4p (also called Ncl1p) provided a structural and evolutionary platform for identification of catalytic residues and modeling of the architecture of the RNA:m5C MTase active site. The analysis led to the identification of two invariant residues that are important for Trm4p activity in addition to the conserved Cys residues in motif IV and motif VI that were previously found to be critical. The newly identified residues include a Lys residue in motif I and an Asp in motif IV. A conserved Gln found in motif X was found to be dispensable for MTase activity. Locations of essential residues in the model of Trm4p are in very good agreement with the X-ray structure of an RNA:m5C MTase homolog PH1374. Theoretical and experimental analyses revealed that RNA:m5C MTases share a number of features with either RNA:m5U MTases or DNA:m5C MTases, which suggested a tentative phylogenetic model of relationships between these three classes of 5-methylpyrimidine MTases. We infer that RNA:m5C MTases evolved from RNA:m5U MTases by acquiring an additional Cys residue in motif IV, which was adapted to function as the nucleophilic catalyst only later in DNA:m5C MTases, accompanied by loss of the original Cys from motif VI, transfer of a conserved carboxylate from motif IV to motif VI and sequence permutation.     

Affinity chromatography of Escherichia coli (m5U54)-methyltransferase on tRNA-agarose.

tRNA-agarose was prepared by condensing periodate-oxidized tRNA to an agarose matrix containing hydrazide functional groups. The tRNA-agarose was used to take partially purified tRNA (m5U54)-methyltransferase to homogeneity. The method is simple and reproducible and gives high yields.     

Mechanism of N-methylation by the tRNA m1G37 methyltransferase Trm5

Trm5 is a eukaryal and archaeal tRNA methyltransferase that catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to the N(1) position of G37 directly 3' to the anticodon. While the biological role of m(1)G37 in enhancing translational fidelity is well established, the catalytic mechanism of Trm5 has remained obscure. To address the mechanism of Trm5 and more broadly the mechanism of N-methylation to nucleobases, we examined the pH-activity profile of an archaeal Trm5 enzyme, and performed structure-guided mutational analysis. The data reveal a marked dependence of enzyme-catalyzed methyl transfer on hydrogen ion equilibria: the single-turnover rate constant for methylation increases by one order of magnitude from pH 6.0 to reach a plateau at pH 7.0. This suggests a mechanism involving proton transfer from G37 as the key element in catalysis. Consideration of the kinetic data in light of the Trm5-tRNA-AdoMet ternary cocrystal structure, determined in a precatalytic conformation, suggests that proton transfer is associated with an induced fit rearrangement of the complex that precedes formation of the reactive configuration in the active site. Key roles for the conserved R145 side chain in stabilizing a proposed oxyanion at G37-O(6), and for E185 as a general base to accept the proton from G37-N(1), are suggested based on the mutational analysis.     

A single methyltransferase YefA (RlmCD) catalyses both m5U747 and m5U1939 modifications in Bacillus subtilis 23S rRNA

Methyltransferases that use S-adenosylmethionine (AdoMet) as a cofactor to catalyse 5-methyl uridine (m(5)U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, and are also found in certain Archaea. These enzymes belong to the COG2265 cluster, and the Gram-negative bacterium Escherichia coli possesses three paralogues. These comprise the methyltransferases TrmA that targets U54 in tRNAs, RlmC that modifies U747 in 23S rRNA and RlmD that is specific for U1939 in 23S rRNA. The tRNAs and rRNAs of the Gram-positive bacterium Bacillus subtilis have the same three m(5)U modifications. However, as previously shown, the m(5)U54 modification in B. subtilis tRNAs is catalysed in a fundamentally different manner by the folate-dependent enzyme TrmFO, which is unrelated to the E. coli TrmA. Here, we show that methylation of U747 and U1939 in B. subtilis rRNA is catalysed by a single enzyme, YefA that is a COG2265 member. A recombinant version of YefA functions in an E. coli m(5)U-null mutant adding the same two rRNA methylations. The findings suggest that during evolution, COG2265 enzymes have undergone a series of changes in target specificity and that YefA is closer to an archetypical m(5)U methyltransferase. To reflect its dual specificity, YefA is renamed RlmCD.     

Identification of catalytic residues in the As(III) S-adenosylmethionine methyltransferase

The enzyme As(III) S-adenosylmethionine methyltransferase (EC 2.1.1.137) (ArsM or AS3MT) is found in members of every kingdom, from bacteria to humans. In these enzymes, there are three conserved cysteine residues at positions 72, 174, and 224 in the CmArsM orthologue from the thermophilic eukaryotic alga Cyanidioschyzon sp. 5508. Substitution of any of the three led to loss of As(III) methylation. In contrast, a C72A mutant still methylated trivalent methylarsenite [MAs(III)]. Protein fluorescence of a single-tryptophan mutant reported binding of As(III) or MAs(III). As(GS)(3) and MAs(GS)(2) bound significantly faster than As(III), suggesting that the glutathionylated arsenicals are preferred substrates for the enzyme. Protein fluorescence also reported binding of Sb(III), and the purified enzyme methylated and volatilized Sb(III). The results suggest that all three cysteine residues are necessary for the first step in the reaction, As(III) methylation, but that only Cys174 and Cys224 are required for the second step, methylation of MAs(III) to dimethylarsenite [DMAs(III)]. The rate-limiting step was identified as the conversion of DMAs(III) to trimethylarsine, and DMAs(III) accumulates as the principal product.     

Structural alterations of the tRNA(m1G37)methyltransferase from Salmonella typhimurium affect tRNA substrate specificity

In Salmonella typhimurium, the tRNA(m1G37)methyltransferase (the product of the trmD gene) catalyzes the formation of m1G37, which is present adjacent and 3' of the anticodon (position 37) in seven tRNA species, two of which are tRNA(Pro)CGG and tRN(Pro)GGG. These two tRNA species also exist as +1 frameshift suppressor sufA6 and sufB2, respectively, both having an extra G in the anticodon loop next to and 3' of m1G37. The wild-type form of the tRNA(m1G37)methyltransferase efficiently methylates these mutant tRNAs. We have characterized one class of mutant forms of the tRNA(m1G37)methyltransferase that does not methylate the sufA6 tRNA and thereby induce extensive frameshifting resulting in a nonviable cell. Accordingly, pseudorevertants of strains containing such a mutated trmD allele in conjunction with the sufA6 allele had reduced frameshifting activity caused by either a 9-nt duplication in the sufA6tRNA or a deletion of its structural gene, or by an increased level of m1G37 in the sufA6tRNA. However, the sufB2 tRNA as well as the wild-type counterparts of these two tRNAs are efficiently methylated by this class of structural altered tRNA(m1G37)methyltransferase. Two other mutations (trmD3, trmD10) were found to reduce the methylation of all potential tRNA substrates and therefore primarily affect the catalytic activity of the enzyme. We conclude that all mutations except two (trmD3 and trmD10) do not primarily affect the catalytic activity, but rather the substrate specificity of the tRNA, because, unlike the wild-type form of the enzyme, they recognize and methylate the wild-type but not an altered form of a tRNA. Moreover, we show that the TrmD peptide is present in catalytic excess in the cell.     

A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase

The modified nucleoside 1-methyladenosine (m¹A) is found in the T-loop of many tRNAs from organisms belonging to the three domains of life (Eukaryota, Bacteria, Archaea). In the T-loop of eukaryotic and bacterial tRNAs, m¹A is present at position 58, whereas in archaeal tRNAs it is present at position(s) 58 and/or 57, m¹A57 being the obligatory intermediate in the biosynthesis of 1-methylinosine (m¹I57). In yeast, the formation of m¹A58 is catalysed by the essential tRNA (m¹A58) methyltransferase (MTase), a tetrameric enzyme that is composed of two types of subunits (Gcd14p and Gcd10p), whereas in the bacterium Thermus thermophilus the enzyme is a homotetramer of the TrmI polypeptide. Here, we report that the TrmI enzyme from the archaeon Pyrococcus abyssi is also a homotetramer. However, unlike the bacterial site-specific TrmI MTase, the P.abyssi enzyme is region-specific and catalyses the formation of m¹A at two adjacent positions (57 and 58) in the T-loop of certain tRNAs. The stabilisation of P.abyssi TrmI at extreme temperatures involves intersubunit disulphide bridges that reinforce the tetrameric oligomerisation, as revealed by biochemical and crystallographic evidences. The origin and evolution of m¹A MTases is discussed in the context of different hypotheses of the tree of life.